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B Kinase, and Insulin Signaling in Human Omental Versus Subcutaneous Adipose Tissue in ObesityDepartment of Clinical Biochemistry (N.B., K.D., T.T., S.O., R.P., A.R.), The National Institute of Biotechnology in the Negev (S.O.), and The S. Daniel Abraham Center of Health and Nutrition (A.R.), Ben-Gurion University of the Negev, Beer-Sheva 84103, Israel; Soroka Medical Center (I.H.-B., I.F.L., T.M.-Z., E.A.), Beer-Sheva 84101, Israel; and Medical Department III and Interdisciplinary Centre for Clinical Research (M.B., M.S.), University of Leipzig, Leipzig D-04103, Germany
Address all correspondence and requests for reprints to: Assaf Rudich, M.D., Ph.D., Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84103, Israel. E-mail: rudich{at}bgu.ac.il.
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Abstract |
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B kinase (IKK) were suggested to link various conditions thought to develop in adipose tissue in obesity (oxidative, endoplasmic reticulum stress, inflammation) with insulin resistance. Yet whether in obesity these kinases are affected in a fat-depot-differential manner is unknown. We assessed the expression and phosphorylation of these kinases in paired omental and abdominal-sc fat biopsies from 48 severely obese women (body mass index > 32 kg/m2). Protein and mRNAs of p38MAPK, ERK, c-Jun kinase-1, and IKKß were increased 1.5–2.5-fold in omental vs. sc fat. The phosphorylated (activated) forms of these kinases were also increased to similar magnitudes as the total expression. However, phosphorylation of insulin receptor substrate-1 on Ser312 (equivalent of murine Ser307) was not increased in omental, compared with sc, fat. Consistently, fat tissue fragments stimulated with insulin demonstrated that tyrosine phosphorylation and signal transduction to Akt/protein kinase B in omental fat was not inferior to that observable in sc fat. Comparison with lean women (body mass index 23.2 ± 2.9 kg/m2) revealed similar ERK2 and IKKß expression and phosphorylation in both fat depots. However, as compared with lean controls, obese women exhibited 480 and 270% higher amount of the phosphorylated forms of p38MAPK and c-Jun kinase, respectively, in omental, but not sc, fat, and this expression level correlated with clinical parameters of glycemia and insulin sensitivity. Increased expression of stress-activated kinases and IKK and their phosphorylated forms in omental fat occurs in obesity, potentially contributing to differential roles of omental and sc fat in the pathophysiology of obesity. |
Introduction |
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B kinase (IKK) (2, 4, 5, 6). JNK is a MAPK best linked to insulin resistance by the demonstration that it can phosphorylate the insulin receptor substrate 1 (IRS1) on Ser307 (or the equivalent Ser312 in humans), thereby rendering IRS1 less amenable to become phosphorylated on tyrosine residues required to propagate the signal to downstream effectors such as protein kinase B (PKB)/Akt (3). IKK can also phosphorylate IRS1 directly (7). Yet its best known function is to phosphorylate the inhibitory unit of nuclear factor-B, subjecting it to enhanced proteasomal degradation, thereby allowing the nuclear translocation of the p65-p55 complex (8). This in turn regulates the transcription of an array of genes, many of which have been implicated in the induction of insulin resistance, in part by increasing serine phosphorylation of IRS1 (3, 9). In addition, increased activation of p38MAPK has been suggested to contribute to adipocyte insulin resistance, mainly by mediating regulation of genes like GLUT4 and the phosphoinositide phosphatase and tensin homolog deleted from chromosome 10 (10, 11, 12). The salient feature of obesity is the excessive accumulation of adipose tissue. Because fat tissue has now been shown to play an active role in regulating whole-body fuel metabolism, understanding the mechanisms by which obesity alters adipose tissue biology is of major interest. Fat tissue in obesity has been shown to exhibit a chronic inflammatory state (13) and is exposed to increased oxidative as well as ER stress (14, 15), all of which are strong inducers of stress signaling. Remarkably, adipose tissue is not uniform in its involvement in the induction of insulin resistance, and different regional depots may render fat accumulation less or more pathogenic. For instance, intraabdominal (or central) adiposity is more strongly associated with the development of comorbid states of obesity, including cardiovascular disease and diabetes, compared with accumulation of sc (peripheral) fat (16, 17, 18). The reasons for this differential depot-specific effect may be a combination of anatomical factors (venous drainage of intraabdominal fat to the liver vs. systemic drainage for the sc fat) as well as inherent metabolic and endocrine differences (19). As an example of the latter, omental fat appears to exhibit more features of inflammation (macrophage infiltration) than sc fat (20), secretes different levels of adipokines and depot-specific factors (21), and displays different responsiveness to insulin and lipolytic stimuli (22, 23) (although not all studies agree on the details).
Here we set up to assess whether human obesity is associated with depot-specific alterations in MAPKs and IKK and, if so, whether these changes are coupled to differences in responsiveness of the insulin signaling machinery.
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Research Design and Methods |
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For comparison with lean controls, six women undergoing explorative laparoscopic or open-abdominal surgery at the Soroka University Medical Center were included. These women were 39.7 ± 12.5 yr old (range 22–59, P = 0.210, compared with the obese cohort), had a mean BMI of 23.2 ± 2.9 kg/m2 (19.0–26.0, P < 0.0001, compared with the obese cohort), and had fasting plasma glucose of 92.0 ± 5.8 mg/dl (83–101, P = 0.468, compared with the obese group).
Adipose tissue lysates
For preparing adipose tissue lysates, omental (OM) and abdominal-sc adipose tissue biopsies were delivered on ice to the laboratory, in which they were rinsed in saline and powdered after freezing in liquid nitrogen. Lysates were prepared using a glass homogenizer, with lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% (vol/vol) Nonidet P-40, 0.25% (wt/vol) sodium deoxycholate, 10 mM sodium ß-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM EGTA, 1 mM sodium vanadate, 1 mM NaF], supplemented with protease inhibitor cocktail (1:1000 dilution; Sigma, St. Louis, MO).
Western blot analysis
Lysates (30 µg per lane, and 60 µg for pS312-IRS1) were separated on 10% polyacrylamide gels, and blotting was performed as previously described (24). The following antibodies were used for immunodetection: anti-Akt/PKB, pS473-Akt/PKB, pT183/Y185-JNK, pS312(murine S307)-IRS1, pT202/Y204-ERK, and pT180/Y182-p38MAPK were from Cell Signaling Technology (Danvers, MA). Antibodies raised against pS181-IKKß, ERK2, p38MAPK, and JNK1 were form Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against IRS1 and IKKß were from Upstate Cell Signaling Solutions (Lake Placid, NY). Monoclonal antibody raised against ß-actin was from Sigma. Each antibody was used according to the manufacturer’s instructions, except for anti-pS312-IRS1, which was used at a dilution of 1:750. Densitometry analysis was used as previously described (24) on scanned films with exposure at the linear range of the signal. In many occasions Western blot analyses were repeated on the same samples two to four times, yielding a mean coefficient of variance of 24.7 ± 4.3%. The mean value of such multiple runs was used for quantification. Equal protein loading was confirmed by blotting samples with anti-ß-actin antibody.
Quantitative real-time PCR
P38MAPK
, ERK, JNK1, IKKß, and IRS1 mRNA expression were determined by quantitative real-time RT-PCR in a fluorescent temperature cycler (Taqman; Applied Biosystems, Darmstadt, Germany). Total RNA was extracted from paired OM and sc adipose tissue samples using RNeasy minikit (including the RNase-free DNase set; QIAGEN, Hilden, Germany), and 1 µg RNA was reverse transcribed with standard reagents (Life Technologies, Grand Island, NY). Expression of each transcript was quantified relative to 18s rRNA gene expression using the second derivative maximum method of the Taqman software (Applied Biosystems, Darmstadt, Germany), as previously described (25, 26). The following primers pairs were used: human ERK, 5'-gctcaaccacattctgggcatc-3' (sense) and 5'-tttctaacagtctggcgggagagg-3' (antisense); human IRS1, 5'-agtcccagcaccaacagaac-3' (sense) and 5'-tcatccgaggagatgaaacc-3' (antisense); human IKKß, 5'-ccggaagtacctgaaccagttt-3' (sense) and 5'-ggacgatgttttctggctttaga-3' (antisense). Human p38MAPK, JNK1, and 18S rRNA expression was measured with a premixed assay on demand (PE Biosystems, Weiterstadt, Germany).
Insulin action in adipose tissue explants
For assessment of insulin signaling in tissue explants, fat tissue biopsies were carefully minced into small tissue fragments of approximately 2–3 mm3 and incubated for 1 h in a CO2 incubator (37 C, 5% CO2) in DMEM supplemented with 2 mM glutamine, 1% (vol/vol) antibiotic solution (all from Biological Industries, Beit-Haeemek, Israel), and with 0.1% (wt/vol) BSA (RIA grade; Sigma). Thereafter tissue fragments were incubated for an additional 10 min in the same medium containing (or not) 100 nM human recombinant insulin (Novo Nordisk, Copenhagen, Denmark). After insulin stimulation, tissue fragmented were collected, and lysates were immediately prepared as described above.
Statistical analyses
For comparing protein expression or mRNA content in OM vs. sc adipose tissue, paired t test was used. When clinical characteristics or the protein expression was compared between lean controls and obese patients, t test was used. For correlational analyses, we log transformed parameters that were not normally distributed and used the nonparametric Spearman test. P = 0.05 was considered the limit for statistical significance.
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Results |
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) to assess the amount and estimate activation of stress kinases of the MAPK family and IKKß, in both fat depots. Representative blots of paired samples of two women representing the interindividual variability of the cohort demonstrate increased protein expression of p38MAPK, ERK2, JNK, and IKKß in OM, compared with sc, fat (Fig. 1A). Quantitative densitometry analysis demonstrate statistically significant mean 1.5–2.5-fold higher expression of the four kinases in the intraabdominal fat depot, whereas the relative amount of ß-actin was unaltered (Fig. 1B). To further determine whether the increased kinases expression in OM fat was the result of differential regulation at the mRNA level, quantitative real-time PCR was performed, and the ratio between each transcript and 18S rRNA was calculated. The relative abundance of p38MAPK and ERK was nearly an order of magnitude higher than that of JNK1 and IKKß (Fig. 2). Nevertheless, in agreement with the protein expression data, a significantly increased mRNA content in OM fat, compared with sc fat, was observed for each of these kinases. We did not detect statistically significant differences in stress kinase expression in either depot between the subgroups of severely obese diabetic and nondiabetic patients.
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). Quantitative densitometry analysis revealed again a mean 1.5–2.5-fold higher level of activated kinases in OM vs. sc fat, being most pronounced for p38MAPK and statistically significant also for JNK and IKKß (Fig. 3B). To estimate whether OM fat exhibited activation of the kinases beyond the increase in total kinase expression, the OM to sc ratio of phospho to total kinases was calculated. This ratio was found to be 1.08 ± 0.14, 1.16 ± 0.20, 1.00 ± 0.20, and 1.14 ± 0.19 for p38MAPK, ERK2, JNK1, and IKKß, respectively. These results indicated that in severely obese women, there is no activation of the various kinases beyond the change in expression level between OM and sc fat.
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, upper blot). Consistently, mRNA levels of IRS1 tended to be increased in OM, compared with sc fat (IRS1mRNA to 18S rRNA ratio was 36.7 ± 10.3 and 24.0 ± 6.4 in OM and sc, fat, respectively, n = 13, P = 0.143). Interestingly, in contrast to the higher level of total IRS1 expression, the amount of IRS1 phosphorylated on Ser312 was comparable between the two fat depots (Fig. 4, lower blot). These findings suggest that the percent of IRS1 molecules that are phosphorylated on Ser312 was in fact lower in OM, compared with sc, fat of severely obese women.
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). The intensity of these two bands was not lower in OM, compared with sc, tissue fragments (n = 8). Acute insulin stimulation was also shown to increase phosphorylation of IRS1 molecules on Ser312 (or murine Ser307) (30, 31). Consistent with the findings in frozen samples, the degree of phosphorylation on this residue in the nonstimulated state was similar between OM and sc samples (Fig. 5B). Yet insulin acutely increased Ser312 of IRS1, particularly in the OM fat. Further downstream in the insulin signaling cascade, OM fat had 1.9 ± 0.2-fold higher levels of Akt/PKB protein content, compared with sc fat (Fig. 5C, middle panel). Insulin-induced phosphorylation of Akt/PKB was assessed using antiphosphoSer473 Akt/PKB antibodies (Fig. 5C, upper blot and graph). The phospho-Akt/PKB band in insulin stimulated OM fat was 1.7 ± 0.1-fold higher than that observed in SC fat, similar to the difference in total Akt/PKB content. Hence, despite increased stress kinase expression in OM fat of obese women (Figs. 1–3), IRS1 Ser312 phosphorylation and signal transmission capacity to Akt/PKB were not decreased compared with sc fat.
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). In contrast, OM fat exhibited differences between lean and obese women in p38MAPK and JNK but not ERK or IKKß. The total amount of p38 MAPK and of JNK1 tended to be higher in OM fat of obese women by 176 and 155%, respectively (Fig. 6, A and C). Remarkably, this difference was more pronounced in the phosphorylated form of both proteins, amounting to a 480 and 270% difference for phosphorylated (p) p38MAPK and p-JNK1, respectively. The mean phospho to total ratio in OM fat was increased by 2.2- and 2.0-fold in the obese compared with lean controls for p38MAPK and JNK, respectively. No similar activation was observed for these kinases in the sc depot. Furthermore, using correlation analysis, OM p-p38MAPK and to a lesser degree p-JNK1, correlated with clinical parameters (Table 2). We did not detect similar correlations with these proteins in the sc depot. Thus, OM fat appears to exhibit increased stress signaling in obesity.
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Discussion |
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Does obesity differentially activate stress signaling in intraabdominal fat?
Although obesity has been suggested to induce oxidative stress, ER stress, and inflammation specifically in adipose tissue (13, 14, 15), all of which are known activators of stress signaling, whether this occurs in a fat depot-differential manner is largely unknown. By comparing kinases expression and phosphorylation in paired OM and sc fat samples of six lean and six obese women, we demonstrate increased stress signaling involving p38MAPK and JNK in OM but not sc fat in the obese women (Fig. 6). No similar differences between obese and lean women were observed in ERK or IKK signaling. This analysis involved a relatively small number of lean vs. severely obese women, potentially limiting our capacity to generalize our findings to males or milder, more common forms of obesity. Nevertheless, despite these limitations and the observational nature of the study, the similar pattern observed with p38MAPK and JNK, distinct from that seen with ERK and IKK, is remarkable. Of the MAPKs, p38MAPK and JNK are preferentially activated by various extracellular and intracellular stresses, whereas ERKs play a central role in cell survival and mitogenic signaling. Although p38MAPK and JNK are directly activated by distinct MKKs, i.e. MKK3/6 and MKK4/7, respectively, these can be activated by a common MKK kinase, apoptosis signal-regulating kinase 1 (33). The latter kinase is rather selectively activated by the inflammatory cytokine TNF, by hydrogen peroxide, and by ER stress, leading via the respective MKK, to the activation of p38MAPK and JNK (reviewed in Ref. 34). Thus, our finding support the notion that obesity induces a more pronounced stress response in OM fat than sc fat, the precise cause of which remains to be established. Interestingly, a recent study demonstrated that obesity is associated with increased macrophage infiltration into OM, compared with sc, fat (20), findings we confirmed in our cohort (40). Moreover, this inflammatory response of OM fat, but not sc, correlated with comorbid states of obesity (20 , 40). Intriguingly, apoptosis signal-regulating kinase 1 was recently suggested to mediate the induction of insulin resistance in response to TNF through mitochondrial oxidants generation (35). Future studies should assess whether in obesity, macrophage infiltration into OM and stress signaling activation in this tissue are two parallel and unrelated phenomena or are interrelated.
Stress kinases in sc vs. OM fat in obesity
In an established obese state, our cohort of severely obese women demonstrates that OM fat expresses higher levels than sc fat of the four kinases measured, at both the protein and mRNA levels (Figs. 1 and 2, respectively). [This was also evident in the subgroup of eight severely obese women, compared with the lean controls (Fig. 6)]. Both MAPKs and IKK are largely thought to be regulated through phosphorylation-mediated cascades rather than by changes in gene expression and protein content. Therefore, the apparent expression differences in the stress kinases between OM and sc fat likely represent intrinsic heterogeneity between the two fat tissue depots. This notion is consistent with the finding in humans using a gene-array approach, of genetically programmed developmental differences between sc and intraabdominal fat depots (25). Intriguingly, the level of expression of some of the differentially expressed genes correlates with BMI, consistent with the aforementioned differences in the expression of stress kinases between lean and obese subjects (Fig. 6). It is noteworthy that the level of activated stress kinases was increased in OM, compared with sc, to the same degree as the increase in total protein expression (Fig. 3). Although this finding does not support differentially increased activity of the upstream MKKs between the two depots in the severely obese state, downstream substrates of the various stress kinases may still be subjected to increased stress signaling input.
Stress kinases and insulin signaling in adipose tissue in obesity
One suggested consequence of increased MAPK and/or IKK input is an impingement on the insulin signaling cascade. A potential mechanism by which this may occur is the phosphorylation of IRS1 on specific serine residues, most studied of which is Ser307 (the murine equivalent of human Ser312) (29, 30, 31). IRS1 molecules phosphorylated on this Ser residue may be a poor substrate for the insulin receptor tyrosine kinase and hence poorly propagate the signal to phosphatidylinositol 3-kinase- and Akt/PKB-required steps for the metabolic actions of insulin in adipose tissue. Differences in insulin action between OM and sc fat have been documented: isolated OM adipocytes exhibited reduced antilipolytic sensitivity and responsiveness to insulin that was attributed to decreased proximal insulin signaling events (insulin receptor tyrosine kinase activity) (22, 36). However, other studies that assessed insulin signaling in adipose tissue in vivo reported higher, although more transient, insulin signaling events in OM, compared with sc, fat in lean patients (23). Two additional studies that used isolated adipocytes from patients with a large range of BMIs showed increased insulin responsiveness (of glucose uptake) in OM vs. sc (37, 38) but marked attenuation of this response by obesity, particularly if centrally distributed (38).
Both technical differences (the use of isolated adipocytes, in vivo biopsies or tissue fragment explants) and patient selection (dissecting out the effects of obesity from the intrinsic differences between OM and sc) may impose a major challenge in reconciling these studies. Yet the present study seems to be more consistent with the notion that even in severely obese women, the responsiveness of OM adipose tissue fragments to insulin is greater (or at least comparable with) that of sc, at least to the level of Akt/PKB activation. Consistently, IRS1 Ser312 phosphorylation was not increased in OM, compared with sc, tissue of severely obese women, despite the mild increase in kinase expression, particularly JNK and IKK, that are known to phosphorylate Ser312 directly or indirectly (3). A potential explanation for this may be that OM fat also exhibits increased IRS1 Ser312 phosphatase activity. Nevertheless, because in obesity adipose tissue is known to lose metabolic insulin responsiveness, it remains to be assessed whether in the intraabdominal fat depot this occurs through signaling defects at the insulin receptor-IRS1 level, potentially involving p38MAPK and JNK activation, or at more distal locations (39). Alternatively, it is possible that although central obesity is closely linked to insulin resistance, insulin signaling at the intraabdominal fat per se is rather maintained. If so, the role of increased visceral fat in the induction of total body insulin resistance may largely reflect its interorgan cross-talk mediated by adipocytokines and other secreted products. The role of MAPKs and/or IKK in mediating these functions may unravel novel intervention strategies for preserving the endocrine-metabolic function of severely obese individuals.
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Acknowledgments |
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We are indebted to Dr. Tatyana Shuster for devoted assistance in patients recruitment and Professor Arnon Wiznitzer for the recruitment of lean controls.
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Footnotes |
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Disclosure Summary: The authors have nothing to disclose.
First Published Online February 22, 2007
Abbreviations: BMI, Body mass index; ER, endoplasmic reticulum; IKK, inhibitory-B kinase; IRS1, insulin receptor substrate 1; JNK, c-Jun kinase; MKK, MAPK kinase; OM, omental; p, phosphorylated; PKB, protein kinase B.
Received October 6, 2006.
Accepted for publication February 14, 2007.
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B/NF-B axis in obesity- and diet-induced insulin resistance. Int J Obes Relat Metab Disord 27(Suppl 3):S49–S52B kinase complex. J Biol Chem 277:48115–48121B system: a treasure trove for drug development. Nat Rev Drug Discov 3:17–26[CrossRef][Medline]B kinase activity through IKKß subunit phosphorylation. Science 284:309–313 stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest 107:181–189[CrossRef][Medline]This article has been cited by other articles:
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2–3 mm3) fragments and incubated ex vivo in culture medium and then incubated without or with 100 nM insulin for 10 min, after which tissue lysates were subjected to Western blot analyses using antiphosphotyrosine antibody (A), anti-pS312 IRS1 antibody (B), or antibodies directed against pS473-Akt/PKB, total Akt/PKB, or ß-actin (C). Shown are blots representative of samples from a total of eight severely obese women, yielding similar results.